Ophrys holubyana Andrasovszky (Orchidaceae), distributed in the Carpatho-Pannonian region, is generally believed to be of hybrid origin. Although its hybrid origin is broadly accepted by many authors, no molecular evidence has been found to support the hypothesis. The nuclear ribosomal DNA internal transcribed spacer (nrDNA ITS) region was sequenced from Ophrys holubyana, and from the presumed progenitor taxa: Ophrys fuciflora (Cr.) Rchb. and Ophrys bicornis Sadler ex Nendtvich. Nearly all the known populations in the Carpatho-Pannonian region were sampled, and the first data on the nrDNA ITS sequence of O. holubyana and O. bicornis are presented. Aligning the ITS sequences revealed no differences among the ten samples. After cloning the amplified ITS regions, eight discrete ITS paralogs with regularly appearing nucleotide differences could be partitioned, differing in only 6 base pairs. Paralog sequences were detected not only in O. holubyana but also in some populations of the two parent species, suggesting that O. fuciflora and O. bicornis in the Carpatho-Pannonian region are also partially of hybrid origin themselves, or influenced by introgression. The study suggests that nrlTS regions can be generally useful in the study of Ophrys systematics and phylogeography and for analyzing the hybrid zones of related Ophrys species groups.
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